Maize is the number one crop in the US. It is used as food for humans and animals and as a source of many compounds of industrial importance. Ethanol for industry and fuel is derived from maize starch. The leftover debris (leaves and stems, called stover) from the annual maize crop represents a huge potential source of organic material for the production of industrial chemicals and fuels.

Development of maize plants with modified cell walls could have many benefits. For example, maize cell walls that were more easily digested by cattle and swine would result in increased feed efficiency, resulting in reduced production costs for farmers and reduced environmental contamination by manure.

Maize is an excellent tool for basic research.  (1) It is easy to grow in large quantities in the laboratory. (2) It is a large plant, which facilitates the harvest of plant material. This is particularly important for biochemical studies such as enzyme purification, where amount of starting material is often limiting. (3) Its cell walls have been extensively studied (e.g., Kato and Nevins, 1984; Carpita et al., 2001). (4) It is an excellent plant for genetic studies. Many tools, such as extensive linkage maps and a transposon tagging system, are available for making mutants and isolating genes (http://www.agron.missouri.edu/). Extensive cDNA databases and >300 MB of gene-rich genomic sequence are now available. It is also possible to use synteny (=genetic co-linearity) between maize and rice to identify maize genes of interest (http://rgp.dna.affrc.go.jp/).

The hemicelluloses of plants in the cereal family (Poaceae, formerly Gramineae) are distinct from those of dicots and even other monocots. Many of the most important food crops are cereals, including rice, maize, wheat, oats, barley, and sorghum. Compared to dicots, cereal cell walls contain larger amounts of glucuronoarabinoxylan (GAX), smaller amounts of pectin, and a particular hemicellulose, called mixed-linked gluan (MLG) or beta-glucan, that is completely absent from other plant species. MLG is a polymer of glucan with beta1,3- and beta1,4-linkages in the ration of ~1:3 (Gibeaut and Carpita, 1993). MLG is particularly abundant in rapidly elongating tissues, is subject to autolysis by endogenous wall enzymes, and has a high turnover rate compared to other wall polysaccharides (Carpita, 1996). Cereals also contain levels of xyloglucan comparable to dicots, although it differs in structure and chemical properties (Hayashi, 1989).

Young etiolated maize seedlings are an excellent tissue for biochemical work. The anatomy of an etiolated maize seedling is shown in Fig. 1. We are using the mesocotyl as an experimental system. Its advantages include relatively large size and fast growth. It is a solid tissue and its growth is highly polar, i.e., like the root, cell divisions occur at the apex and elongation occurs mainly by cell expansion.

The mesocotyl is a transient organ whose function is to position the critical shoot meristem (at the coleoptilar node) at ground level. Growth of the mesocotyl is inhibited by light (Fig. 2). This response is mediated by phytochrome (i.e., red light is the most effective wavelength, and the response is reversible by far-red light). The phytochrome effect is mediated by auxin; light causes auxin levels in the mesocotyl to decrease, which in turn causes a reduction in cell explansion and thus growth. 

In the mesocotyl, an enzyme called glucan synthase I (GSI) is down-regulated by light. The drop in GSI activity occurs over a period of about 8 hr after light treatment, suggesting that regulation is at the transcriptional level (Walton and Ray, 1982a). Exogenous auxin (IAA) prevents the light decline but does not reverse it (Walton and Ray, 1982b). GSI is in the Golgi (Fig. 3). Digestion of the product of GSI with cellulase yields cellobiose (= dimer of beta1,4-linked glucose) (JD Walton and H Van Erp, unpublished results). The product of GSI is presumably not cellulose (which is made at the plasma membrane) and our current assumption is that this material is the backbone of  xyloglucan. Our strategy is to exploit the light-regulation of GSI to help us identify the responsible enzyme and hence gene.

Regulation of Golgi glucan synthase I by light is specific. GSI in the shoot is not affected by light, and xylan synthase (defined as incorporation of UDP-Xyl into ethanol-insoluble product) is also not affected by light in either the mesocotyl or the shoot.

Figure 1. The etiolated maize seedling.

Figure 2. Light inhibits growth of the maize mesocotyl. Plants on the right were grown in complete darkness for 5 d. Plants on the left were exposed to light for 10 min per day.  

Fig. 3. Density fractionation of membranes from dark-grown and light-treated 1-2 cm mesocotyl segments. GSI (Golgi-localized glucan synthase) is assayed at low (~100 nM) UDP-Glu concentration and 30 mM Mg⁺². GSII (plasma membrane-localized glucan [callose] synthase) is assayed at high UDPG (~1 mM) concentration and no exogenous Mg⁺².

Light treatment was 10 min red light 16 hr before harvest. The results show that light causes a large decline in GSI activity but has no effect on either GSII activity or IDPase activity, another Golgi-localized enzyme. Mitochondrial and endoplasmic reticulum markers (cytochrome c oxidase and cytochrome c reductase, respectively) are also not affected by light (not shown). (From Walton and Ray, 1982a.)

References:

Carpita NC (1996) Structure and biogenesis of the cell walls of grasses. Annu Rev Plant Physiol Plant Mol Biol 47:445-476.

Carpita NC, M Defernez, K Findlay, B Wells, DA Shoue, G Catchpole, RH Wilson, MC McCann (2001) Cell wall architecture of the elongating maize coleoptile. Plant Physiol 127: 551-565.

Gibeaut DM, Carpita NC (1993) Synthesis of (1-3), (1-4)-beta-D-glucan in the Golgi apparatus of maize coleoptiles. Proc Natl Acad Sci USA 90:3850-3854.

Hayashi, T (1989) Xyloglucans in the primary cell wall.  Annu Rev Plant Physiol 40:139-168.

Kato Y, Nevins DJ (1984) Enzymic dissociation of Zea shoot cell wall polysaccharides .1. Preliminary characterization of the water-insoluble fraction of Zea shoot cell-walls. Plant Physiol 75: 740-744.

Walton JD, PM Ray (1982a) Inhibition by light of growth and Golgi-localized glucan synthetase activity in the maize mesocotyl. Planta 156:309-313.

Walton JD, PM Ray (1982b) Auxin controls Golgi-localized glucan synthetase in the maize mesocotyl. Planta 156:302-308.

Revised July 29, 2004